Inositol 1,4,5-trisphosphate (InsP3) mediates the release of Ca from intracellular stores by interacting with a family of[unreadable] highly conserved receptors which contain an intrinsic Ca release channel. The InsP3 receptor (InsP3R) shares both[unreadable] structural and sequence similarities with the ryanodine receptor (RyR), the primary Ca release channel of the[unreadable] sarcoplasmic reticulum (SR) in cardiac and skeletal muscle. The type-2 InsP3R is the predominant isoform expressed[unreadable] in cardiac myocytes, but its localization and function in heart have been elusive. In ventricular myocytes the InsP3R2 is[unreadable] targeted to the nuclear envelope and it interacts with Ca-calmodulin dependent protein kinase II (CaMKII). A goal of[unreadable] this project is to identify and characterize proteins interacting with the InsP3R2 expressed in cardiac myocytes that are[unreadable] critical for function and modulation of local Ca signaling events (regarding both regulation of InsP3R by CaMKII and[unreadable] regulation of CaMKII by Ca release from InsP3R). The central focus encompasses detailed study of InsP3R2/CaMKIIdelta interactions and the functional consequences with respect to Ca signaling and nuclear signaling in hypertrophy & heart failure. A second emphasis is the characterization, use and further development of novel FRET-based biosensors for in situ measurement of InsP3 liberation and concentration.[unreadable] There are 5 Specific Aims in this project that will address the InsP3R2/CaMKII interaction and function.[unreadable] 1) Test the hypothesis that the InsP3R2 localized to the nuclear envelope of cardiac myocytes interacts with CaMKII[unreadable] and forms a macromolecular complex.[unreadable] 2) Test the hypothesis that the interaction of CaMKII directly modulates the InsP3R2 Ca release channel properties.[unreadable] 3) Determine the physiological consequences of perturbing or disrupting the InsP3R2-CaMKII interaction.[unreadable] 4) Test the hypothesis that the spatial/temporal patterns of expression levels for the InsP3Rs are altered during ES cell[unreadable] development, hypertrophy and heart failure.[unreadable] 5) Generate a biological sensors to measure the free concentration of InsP3 in an intact cells.[unreadable] These studies will provide considerable new information regarding InsP3R in cardiac myocytes in health and disease.[unreadable] This is especially the case with respect to localization, molecular partners and regulation of function. Additionally, we[unreadable] anticipate that the InsP3-biosensors will become important new tools in the InsP3 field. This work is highly synergistic[unreadable] with all three other projects in the Program Project Grant.[unreadable]